Functional Analysis Of Two AINTEGUMENTA-LIKE And Four CALEOSIN In Lipid Metabolism In Arabidopsis And Brassica Napus L.

Plant oil is the primary high-energy storage material in plant seeds.It has numerous food and industrial uses,and also serves as a crucial source of essential fatty acids for humans.Research on the synthesis and regulation of plant oil has important biological significance as well as significant social and economic implications.The biochemical process of oil biosynthesis in plants has been studied well,but more research is needed to fully understand its regulatory mechanisms.FUSCA3（FUS3）is one of the few positive regulators reported,however,the molecular mechanism of how FUS3 regulates lipid metabolism is still unclear.In this study,we conducted functional research on two AINTEGUMENTA-LIKE genes（AIL6and AIL1）and four CALEOSIN genes（CLO1,CLO2,CLO4,and CLO6）,which can be regulated by FUS3 and highly expressed in seeds,to gain insight into their molecular mechanisms in regulating lipid metabolism by FUS3.The main results of this study are as follows:（1）AIL6,as a downstream regulatory factor of FUS3,affects seed fatty acid metabolism by regulating FAD3 and FAE1,and affects seed dormancy by regulating the biosynthesis and signaling pathways of GA,ABA,and IAA:AIL6 activation by FUS3 was confirmed through induction and dual-luciferase assays;Gene expression analysis showed that the expression levels of AIL6 was gradually increased during seed development;The fatty acid compositions of the seeds of the ail6 mutants were altered,and both Arabidopsis AIL6（At AIL6,abbreviated as AIL6.）and Brassica napus AIL6（Bna AIL6）were able to rescue changes in the fatty acid composition of the ail6 mutant;Overexpression of AIL6 and Bna AIL6 in Arabidopsis easily led to transgenic co-suppression,which was relieved by the rdr6-11 mutation;There is a change in the fatty acid composition in the seeds of Arabidopsis or camelina transgenic lines overexpressing AIL6 with a high expression level,which is opposite to the changes in the ail6mutant.Specifically,there is a significant increase in the content of 18:1 and 18:2,while the content of 18:3 and 20:1 is significantly decreased,especially with the largest changes observed in the content of 18:2 and 18:3.The ratio of 18:2 to 18:3 in the AIL6-overexpressing Arabidopsis seeds increased from 1.5 in the wild type to more than five.Overexpression of AIL6 or Bna AIL6 in camelina increased the ratio of 18:2 to 18:3 from 0.45 in the wild type to more than 1.1 in seeds of OE lines.Transcriptome analysis of developing seeds of the AIL6OE 32 and ail6-1 mutant revealed that AIL6 regulated genes related to lipid metabolism and the changes of fatty acid compositions in AIL6 OE lines may be the results of downregulation of FAD3 and FAE1.Furthermore,ultrahigh expression of AIL6 was found to cause deep seed dormancy,delayed seed germination,and reduced germination rate,with the lowest germination rate as low as 12%.Further measurement of plant hormones in the AIL6 OE lines revealed that the content of GA₄ decreased to below 6%of that in the wild type,while the content of ABA and IAA increased.Exogenous GA₃ treatments in developing seeds or germinated seeds did not effectively rescue the low germination rate or delayed germination.Nicking seed coats or elongation of after-ripening time could partly rescue the low germination rate and lead to the increased germination rate to nearly 80%and 40%,respectively.Furthermore,it was found that the deep dormancy caused by AIL6 overexpression could be effectively released by the GA receptor della quintuple mutant.Overall,these results indicated that AIL6,as a downstream regulatory factor of FUS3,has a role in fatty acid metabolism and seed dormancy.（2）AIL1,as a downstream regulator of FUS3,partially regulates fatty acid metabolism and oil accumulation through LPAT5,and AIL1 actives oil accumulation in vegetable tissues by upregulating the expression of OLE1 and CLO1:In the fus3-T mutant,the expression of AIL1 was downregulated;FUS3 could activate the expression of AIL1;Expression analysis showed that AIL1 was highly expressed in developing seeds,especially in 12 days after flowering seeds;There was no significant change in the fatty acid compositions of the seeds in the ail1 mutants.Changes in the seed fatty acid composition caused by AIL1 overexpression in Arabidopsis or camelina were similar to those caused by overexpression of AIL6,while overexpression of Bna AIL1-1 in rdr6-11 did not result in significant changes in seed fatty acid composition.In addition,it was worth noting that overexpression of AIL1 in Arabidopsis seeds led to an increase in seed oil content.Induction of AIL1 expression in Arabidopsis seedlings resulted in a significant increase in triacylglycerol content in seedlings,and the expression of OLE1 and CLO1 was upregulated.Transcriptome analysis showed that overexpression of AIL1in seeds caused 41 lipid metabolisms differently expressed genes,of which LPAT5 was upregulated.Seeds from LPAT5 OE lines showed the increase of 18:1 content and the decrease of 18:3 content and the increase of seed oil content.AIL1 overexpression in the lpat5 mutant did not show significant differences in seed oil content and resulted in smaller changes in 18:1and 18:3 than AIL1 overexpression in Col-0 background.Taken together,these results suggested that AIL1,as a downstream regulator of FUS3,plays important roles in fatty acid metabolism and oil accumulation.（3）CALEOSINs were functionally redundant in seed fatty acid metabolism and oil accumulation and could affect embryo development:Four caleosin genes CLO1,CLO2,CLO4and CLO6 were highly expressed during seed development;CLO1,the highest expressed CALEOSIN in seeds together with CLO2 were upregulated by FUS3;Four single mutants clo1,clo2,clo4 and clo6 showed similar minor changes in fatty acid compositions in seeds and did not show significant changes in seed oil content;Two double mutants clo1×clo2 and clo1 clo2（created by crossing and CRISPR,respectively）showed significant changes in fatty acid compositions and a 16–23%decrease in seed oil content.In 4CLO RNAi T₁ seeds,the seed oil content decreased by 40%,and the fatty acid compositions further changed.Additionally,the lipid droplets in the embryos became smaller,and the membranes of lipid droplets were thickened.Complementary experiments showed that CLO1 and Bna CLO1 rescued the change of fatty acid compositions and low seed oil contents in clo1 clo2 seeds.Induced expression of CLO1 in Arabidopsis seedlings,leaves and BY2 cells increased the triacylglycerol content.While mutation of the EF-hand motif of CLO1（named CLO1 DD）could also recuse the changes of fatty acid compositions and the decrease of oil content in clo1 clo2 seeds.Furthermore,the seeds of 4CLO RNAi T₁ were shrunken and the germination rate was reduced;Some embryos did not develop into normal cotyledons and hypocotyls or even were undifferentiated spherical embryos;Some germinated seedlings also exhibited abnormal morphology.Transcriptome analysis of clo1 clo2 developing seeds showed that expression levels of genes related to lipid metabolism,seed/embryo development,stress responses and calcium signaling were changed.Together,these results indicated that CALEOSINs play important roles in oil accumulation and embryo development.In this study,we screened and characterized the functions of FUS3 downstream regulators AIL6,AIL1 and seed-expressed caleosins in fatty acid metabolism.The research was conducted using the model plant Arabidopsis,tobacco BY2 cells and the oil crop camelina,and the function of homologous genes in Brassica napus L.was identified.These findings have important implications for future genetic engineering efforts aimed at improving traits such as oil content in crops.The study also revealed the role of AIL6 in seed dormancy,which has potential applications in controlling crop seed germination.